The human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS) through infection of especially helper (CD4+) T cells. Recently, it has been reported that higher levels of cytotoxic lymphocytes (CTLs or CD8+ T cells) which secrete the three cytokines, interferon gamma, interleukin-2 and tumor necrosis factor alpha, confer greater host resistance to HIV, but the mechanism causing this "multi-functional" phenotype remains unknown. Many suffering from AIDS use marijuana (Cannabis sativa) for control of disease symptoms. However, cannabinoids from C. sativa, (e.g., 9-tetrahydrocannabinol (THC)) are known to suppress immune function through presumably cannabinoid receptor 2 (CB2) and cannabinoid receptor 1 (CB1). Thus, benefits of phytocannabinoid-use by HIV patients remain uncertain. More recently, endogenous cannabinoids (endocannabinoids) were discovered. These compounds bind and activate CB1 and CB2 and act through other receptors as well, most notably peroxisome proliferation-activated receptor gamma (PPAR3). The two most characterized endocannabiniods are anandamide (AEA) and 2- arachidonoylglycerol (2-AG). These compounds with their receptors are termed the "endocannabinoid system". Current scientific consensus deems this system plays a role in immune homeostasis by as yet unknown mechanisms. The studies of this proposal seek to help elucidate the mechanism(s) by which the endocannabinoid system maintains immune homeostasis in HIV-infected cells, including modulation of multifunctional T cells. This work, by better defining the physiological role of endocannabinoids in immune regulation, will additionally laid the foundation for future investigations discovering the potential etiology of phytocannabinoid-induced impairment of the endocannabinoid system. For this work, three specific aims will test the hypothesis: Endocannabinoids suppress the anti-viral (HIV-associated gp120-induced) T cell response by impairing CD8+ and/or CD4+ T cell effectors function through a cannabinoid-receptor independent manner. Specific Aim 1 and Specific Aim 2 will determine the time and dose of maximal CD4+ and CD8+ T cell suppression by AEA in wild type C57Bl/6 mice and CB1/CB2 knockout mice, respectively. Specific Aim 3 will examine the relative contributions of CB1, CB2 and PPAR3 signaling in these effects. All experiments will employ an in vitro model of HIV infection, which co-cultures lentivirally transfected cells expressing the HIV envelope protein, gp120, with mouse T cells. Flow cytometry to determine surface marker staining for identification of T cell subpopulation, phases of activation and cytokine production, will be the primary method used. 51Cr-release cell-lysis assays will definitively determine effector CTL activity. As well, ELISPOT and ELISA assays will also quantify cytokine synthesis. PUBLIC HEALTH RELEVANCE: Persons infected with human immunodeficiency virus (HIV) and suffering from acquired immunodeficiency syndrome (AIDS) often utilize marijuana to control disease symptoms. Cannabinoids, the biologically active compounds in marijuana, and endocannabinoids, naturally made compounds in humans that mimic the actions of plant cannabinoids, suppress T cell function, and decrease immune responses. This proposal seeks to determine the role of endocannabinoids in immune suppression against HIV by examining the peak time and concentrations at which they induce T cell suppression in order to better understand the molecular receptors and pathways utilized by cannabinoids and endocannabinoids in regulating anti-HIV T cell responses.